CN100371343C - Molecule marking method for discriminating cytoplasmic male sterile gene of leaf mustard - Google Patents
Molecule marking method for discriminating cytoplasmic male sterile gene of leaf mustard Download PDFInfo
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- CN100371343C CN100371343C CNB2005100503196A CN200510050319A CN100371343C CN 100371343 C CN100371343 C CN 100371343C CN B2005100503196 A CNB2005100503196 A CN B2005100503196A CN 200510050319 A CN200510050319 A CN 200510050319A CN 100371343 C CN100371343 C CN 100371343C
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Abstract
The present invention relates to a molecular marking method for discriminating cytoplasmic male sterile genes of leaf mustard. The method comprises the following steps: extracting a cytoplasmic male sterile line of leaf mustard and backcrossing generation genome DNA thereof; designing a merging primer based on the backcrossing generation genome DNA according to a nap type cabbage type rape cytoplasmic male sterile related gene orf222 and a pol type cabbage type rape cytoplasmic male sterile related gene orf224; carrying out a chain polymerase reaction; detecting a characteristic strip of a sterile molecular marker of an amplification product in the cytoplasmic male sterile line of the leaf mustard and the backcrossing generation thereof. The present invention detects the cytoplasmic male sterile molecular marker of the leaf mustard in the backcrossing generation of the cytoplasmic male sterile line of the leaf mustard for the first time, and establishes a basis for identifying the cytoplasmic male sterile specific germplasm resource of leaf mustard.
Description
Technical field
The present invention relates to a kind of plant molecular marker technology, exactly is a kind of molecule marking method of differentiating leaf mustard cytoplasmic male sterility distinguished germ plasm resource, belongs to technical field of molecular biology.
Background technology
Along with development of molecular biology, molecular marking technique has become assistant breeding and has differentiated the important means of distinguished germ plasm resource, utilizes the molecular marker assisted selection can be early stage as identifying seedling stage in breeding; Be not subjected to the influence of factors such as environment in the chosen process.The molecule marker that crop breeding is commonly used has RFLP, RAPD, SSR and SCAP mark etc.This method adopts the specific molecular marker technology to obtain leaf mustard cytoplasmic male sterility molecule marker, and is used for the Screening and Identification of leaf mustard cytoplasmic male sterility distinguished germ plasm resource.
Cytoplasmic male sterility (cytoplasmic male sterility) is genetic phenomenon commonplace in the plant, in the crop heterosis utilization significant application value is arranged.Because cytoplasmic male sterility can not produce normal function pollen, therefore, utilize male sterile both can save the formality of artificial emasculation, can guarantee the purity of first-filial generation seed again, it is again the good experimental system of research pollen development, plasma inheritance and nucleo-cytoplasmic interreaction simultaneously.Crop in cruciferae exists tangible hybrid vigour, and the production of the first generation of hybrid mainly is to utilize self incompatibility and two kinds of approach of male infertility at present.Cytoplasmic male sterility is because it can obtain the material of 100% sterile degree and sterile rate in theory, therefore, in the utilization of crop in cruciferae heterosis breeding, more wide prospect is arranged, many important cash crop have all selected the good male sterile line of comprehensive proterties as Chinese cabbage, wild cabbage, swede type rape etc. in the Cruciferae.
Leaf mustard is the special product vegetables of China, belong to cress, cultivation is widely arranged on Zhejiang and Sichuan and other places, economic worth is big, be important vegetable for processing, have degradation problem under the serious and quality of virus disease in the production, and heterotic utilization can improve the quality and the raising resistance of existing leaf mustard kind.Because mustard crop self-compatible index is very high, is difficult to utilize self incompatible line to carry out heterotic utilization in the production.Therefore, male sterile application of mustard crop cell matter and theoretical investigation have significance, and adopt cytoplasmic male sterility to obtain hybrid vigour is one of focus of breeding research always.Up to now, in crop in cruciferae, the cytoplasmic male sterile line of having used on producing mainly contains radish cytoplasmic male sterility, Brassica nap type swede type rape cytoplasmic male sterile line and the Brassica pol type swede type rape cytoplasmic male sterile line of Ougra type.Up to the present, the molecule marker report that does not still have cytoplasmic male sterile gene of leaf mustard.
Summary of the invention
The purpose of this invention is to provide the molecule marking method of differentiating cytoplasmic male sterile gene of leaf mustard.
Differentiate the molecule marking method of cytoplasmic male sterile gene of leaf mustard, may further comprise the steps:
Extract leaf mustard cytoplasmic male sterile line and backcross each genomic dna from generation to generation, with it is template, according to nap type swede type rape cytoplasmic male sterility genes involved orf222 and pol type swede type rape cytoplasmic male sterility genes involved orf224 design degenerate primer, carry out the chain polymerization enzyme reaction, amplified production adopts agarose gel electrophoresis in leaf mustard cytoplasmic male sterile line and backcross that each detects the characteristic strip of sterile molecule marker from generation to generation.
Degenerate primer described in the above-mentioned molecule marking method, promptly the nucleic acid molecule of synthetic is:
Forward primer: 5 '-ATGCCTCAACTGGATAAATTCACTT-3 '
Reverse primer: 5 '-TCATCGAAATAGATCRAGKATYTC-3 ', wherein: R is G, A; K is G, T; Y is C, T
The concrete steps of above-mentioned chain polymerization enzyme reaction (PCR) are as follows:
(1) at first following reagent is mixed in together
10 * Taq dna polymerase buffer liquid, 5 microlitres (μ l)
Forward primer (1.25 μ g/l) 0.4 μ l
Reverse primer (1.25 μ g/l) 0.4 μ l
Deoxynucleoside acid mixture (dNTP) 1 μ l
Taq archaeal dna polymerase 0.4 μ l
Aqua sterilisa 41.8 μ l
Cumulative volume 50 μ l
(2) with 94 ℃ of sex change of above-mentioned mixed liquid 3 minutes, enter circulation then: 94 ℃ of sex change 30 seconds, 50 ℃ of annealing 45 seconds, 72 ℃ were extended 1 minute, and 35 circulations were extended 10 minutes for back 72 ℃, an increase band of 663 of result.
(3) amplified production is adopted 1% agarose gel electrophoresis detect
The present invention backcrosses in leaf mustard cytoplasmic male sterile line first, and each detects a molecule marker of leaf mustard cytoplasmic male sterility from generation to generation, lays the foundation for differentiating leaf mustard cytoplasmic male sterility distinguished germ plasm resource.
Description of drawings
Fig. 1 leaf mustard cytoplasmic male sterility molecule marker is in sterile line and the signature in each generation of backcrossing thereof;
1 is the leaf mustard maintenance line among the figure; 2 is sterile source; 3 is sterile source and leaf mustard F
1Generation; 4 is first backcross generation (BC
2); 5 is third backcross generation (BC
3); Arrow is leaf mustard cytoplasmic male sterility molecule marker.
Embodiment
The cytoplasmic male sterile gene of leaf mustard molecule marking method
Leaf mustard cytoplasmic male sterile line (Brassica juncea var.multiceps Tsen etLee) is provided by this laboratory, and material is in Zhejiang University's agricultural and the Vegetable Research Institute plantation of department of horticulture of biotechnology institute.
(1) design of primers: according to Brassica nap type cytoplasmic male sterility genes involved orf222 in the Genebank database and Brassica pol type cytoplasmic male sterility genes involved orf224 (orf222 gene Genebank accession number: U10423, orf224 gene Genebank accession number: S46795 all can find in http://www.ncbi.nlm.nih.gov/) the design degenerate primer.The nucleic acid molecule of synthetic:
Forward primer: 5 '-ATGCCTCAACTGGATAAATTCACTT-3 '
Reverse primer: 5 '-TCATCGAAATAGATCRAGKATYTC-3 ', wherein: R is G, A; K is G, T; Y is C, T
(2) the CTAB method is extracted the total DNA of plant
(a) get the 0.5g tender leaf in mortar, the liquid nitrogen state grinds down, adds 2 * CTAB of 65 ℃ of preheatings of 600 μ l, and (30 μ l mercaptoethanol mixing) transferred in the centrifuge tube, and 65 ℃ are incubated 1-2 hour.
(b) add the equal-volume chloroform: primary isoamyl alcohol (24/1) shakes up gently, and 4 ℃, 13000rpm is centrifugal, 10 minutes.
(c) get supernatant, repeat (2) step once.
(d) get supernatant in another eppendorf pipe, add the equal-volume Virahol, put upside down mixing, left standstill 13000rpm, centrifugal 10 minutes 5 minutes.
(e) remove supernatant, add 700 μ l Wash Buffer rinsing DNA precipitation, centrifugal, repeat once.
(f) add 15 microlitre TE-Rnase, 37 ℃ are incubated 1 hour.
(g) add 2mol/L NaCl 100 μ l+100% ethanol 250 μ l mixings ,-20 ℃ leave standstill 30min, centrifugal 10 minutes of 13000rpm.
(h) remove supernatant, add 500 μ l, 70% ethanol, ethanol is removed in rinsing, repeats once, and normal temperature dries.
(i) add 30-50 μ l ddH20 or TE, Hui Rong ,-20 ℃ of preservations are standby.
Annotate: 2 * CTAB prescription, 200 μ l:
CTAB： 4g
Tris(pH8.0)：?20ml
EDTA： 1.488g
NaCl： 16.352g
TE-RNase:15 μ l TE adds the RNase of 1/100 volume
Wash Buffer:10mmol/L acetate ammonia
76% ethanol
The reagent of (3) chain polymerization enzyme reaction (PCR)
At first following reagent is mixed in together
10 * Taq dna polymerase buffer liquid, 5 microlitres (μ l)
Forward primer (1.25 μ g/l) 0.4 μ l
Reverse primer (1.25 μ g/l) 0.4 μ l
Deoxynucleoside acid mixture (dNTP) 1 μ l
Taq archaeal dna polymerase 0.4 μ l
Aqua sterilisa 41.8 μ l
Cumulative volume 50 μ l
(4) PCR reaction conditions:, enter circulation then with 94 ℃ of sex change of above-mentioned mixed liquid 3 minutes: 94 ℃ of sex change 30 seconds, 50 ℃ of annealing 45 seconds, 72 ℃ were extended 1 minute, and 35 circulations were extended 10 minutes for back 72 ℃.
(5) Markers for Detection: amplified production is at 1% agarose gel electrophoresis, dyeing in the ethidium bromide (ethidiumbromide), ultraviolet lamp is observed down, gel imaging system (U.S. Bio-Rad company product) Taking Pictures recording, the result is in leaf mustard cytoplasmic male sterile line and backcross that each all can detect the existence of this mark, mark shown in Fig. 1 arrow from generation to generation.
Claims (3)
1. the oligonucleotide primer of a synthetic is right, it is characterized in that having the particular sequence of following based composition:
Forward primer: 5 '-ATGCCTCAACTGGATAAATTCACTT-3 '
Reverse primer: 5 '-TCATCGAAATAGATCRAGKATYTC-3 ', wherein: R is G, A; K is G,
T; Y is C, T
2. molecular marker method of differentiating cytoplasmic male sterile gene of leaf mustard, it is characterized in that extracting leaf mustard cytoplasmic male sterile line and backcross each genomic dna from generation to generation, with it is template, oligonucleotide primer according to nap type swede type rape cytoplasmic male sterility genes involved orf222 and the narration of pol type swede type rape cytoplasmic male sterility genes involved orf224 design claim 1 is right, carry out the chain polymerization enzyme reaction, amplified production adopts agarose gel electrophoresis in leaf mustard cytoplasmic male sterile line and backcross that each detects the characteristic strip of sterile molecule marker from generation to generation.
3. method as claimed in claim 2 is characterized in that the concrete steps of chain polymerization enzyme reaction are as follows:
(1) at first following reagent is mixed in together
10 * Taq dna polymerase buffer liquid, 5 μ l
Template DNA 1 μ l
Forward primer (1.25 μ g/l) 0.4 μ l
Reverse primer (1.25 μ g/l) 0.4 μ l
Deoxynucleoside acid mixture (dNTP) 1 μ l
Taq archaeal dna polymerase 0.4 μ l
Aqua sterilisa 41.8 μ l
Cumulative volume 50 μ l
(2) with 94 ℃ of sex change of above-mentioned mixed liquid 3 minutes, enter following circulation then: 94 ℃ of sex change 30 seconds, 50 ℃ of annealing 45 seconds, 72 ℃ were extended 1 minute, and 35 circulations were extended 10 minutes for back 72 ℃,
(3) amplified production is adopted 1% agarose gel electrophoresis detect.
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| CNB2005100503196A CN100371343C (en) | 2005-05-13 | 2005-05-13 | Molecule marking method for discriminating cytoplasmic male sterile gene of leaf mustard |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101017152B (en) * | 2007-02-09 | 2010-07-07 | 南京农业大学 | Rapid identification method for genetic purity of cabbage seed |
| CN101492738B (en) * | 2009-03-09 | 2011-09-21 | 山东省农业科学院蔬菜研究所 | Onion cytoplasmic male sterility SCAR mark and uses thereof |
| CN112725497B (en) * | 2021-01-12 | 2022-04-08 | 华中农业大学 | Molecular marker for identifying stem mustard oxa cytoplasmic male sterility restoring gene and application thereof |
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Non-Patent Citations (1)
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| 胞质雄性不育性在叶用芥菜上的利用. Nikornpun，M.，N.Senapa.中国蔬菜，第1期. 2004 * |
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